PROJECT SUMMARY Recognizing the power of a systems approach to complex disease, during the initial funding period we developed a highly-controlled three color array and analysis platform focused on acquisition of quality data. Our goal is apply these tools to the events leading to autoimmunity using a unique derivative of the BioBreeding (BB) rat model of type 1 diabetes mellitus (T1DM). BB DRlyp/lyp rats are spontaneously diabetic due to a mutation in the Gimap5 gene, rendering them deficient in regulatory T (TREG) cells. DR+/+ rats possess an intact Gimap5 gene and do not develop spontaneous T1DM, but become diabetic upon depletion of TREG cells. T1DM in BB rats also involves a cytotoxic T cells, since their depletion is protective. Our gene expression studies of prediabetic pancreatic lymph nodes (PLN) have shown that mast cells are terminally activated in DRlyp/lyp animals versus negatively regulated in DR+/+ animals; we have confirmed their functional role through preventing T1DM in rats with two different mast cell inhibiting drugs. Thus, we hypothesize that in addition to cytotoxic T effector cells, mast cells play a crucial early role in DRlyp/lyp diabetogenesis and in spite of their disease predisposition, TREG cells are sufficient to prevent T1DM in DR+/+ rats. We further hypothesize that associated with these states are distinct cytokine milieus, since we find incubation of sera derived from human individuals at risk for T1DM or recent onset T1DM with healthy peripheral blood mononuclear cells (PBMC) induce a characteristic gene expression signature that is distinct from normal controls and long standing T1DM. Importantly, we find observe this molecular signature as much as 5 years prior to T1DM onset. The predictable disease course of the BB rat enables longitudinal dissection of the activities of immune cell subpopulations and examining how these activities are manifested in the periphery. Thus we propose to 1) temporally and spatially dissect the activities of antigen presenting, regulatory, and effector cells at defined pre-onset timepoints through gene expression profiling and histological approaches. 2) The sera of WF, DR+/+ and DRlyp/lyp rats will be longitudinally evaluated for factors that induce gene expression in healthy PBMCs. Presence of candidate mediators and disease time point-specific biomarkers will be confirmed. Understanding the relationship between mast cells, T effector, and TREG cells in autoimmunity and has the potential to establish a new paradigm for immune regulation and create new insights to human disease.